Synthesis and computational evaluation of the antioxidant activity of pyrrolo[2,3-b]quinoxaline derivatives

Pyrrolo[2,3-b]quinoxaline derivatives are known to possess antioxidant, anticancer, and antibacterial properties. Here we report the successful synthesis of five derivatives of 3-hydroxy-3-pyrroline-2-one through substitution. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay was employed to evaluate the antioxidant activity of the compounds. Out of these, ethyl 1,2-diphenyl-1H-pyrrolo[2,3-b]quinoxaline-3-carboxylate (3a) demonstrated the greatest potential as a radical scavenger. Thermodynamic and kinetic calculations of the radical scavenging activity indicated that 3a exhibited HO˙ radical scavenging activity with the overall rate constant of 8.56 × 108 M−1 s−1 in pentyl ethanoate; however, it was incapable of scavenging hydroperoxyl radicals in nonpolar media. In non-polar environments, the hydroxyl radical scavenging capability of 3a is fairly similar to that of reference antioxidants such as Trolox, melatonin, indole-3-carbinol, and gallic acid. Hence, in the physiological lipid environment, 3a holds promise as a scavenger of HO˙ radicals.


Experimental and computational methods
2.1.Experimental 2.1.1.Chemicals and experimental methods.All chemicals were purchased from Merck, Acros, or Sigma-Aldrich without further purication.70-230 mesh silica 60 (E.M. Merck) was used as the stationary phase for column chromatography.NMR spectra were acquired on Bruker Avance II+ 600 MHz instruments.The chemical shis (d) were reported in parts per million (ppm) relative to tetramethylsilane (TMS) or the internal (NMR) solvent signals.High-resolution mass spectra (ESIquadrupole) of compounds 3a, 3b and 3e were measured on a quadrupole orthogonal acceleration time-of-ight mass spectrometer (Synapt G2 HDMS, Waters, Milford, MA).In addition, high-resolution mass spectra (ESI-TOF MS/MS) of compounds 3a 0 , 3c and 3d were also recorded on a SCIEX X500 QTOF with an electrospray ionization source in a positive ion mode.Büchi Melting Point B-545 apparatus was used to determine the melting points of all products.
2.1.2.General procedure for the synthesis of pyrrolo[2,3-b] quinoxalines 3a-e.1,5-Disubstituted-4-ethoxycarbonyl-3hydroxy-3-pyrroline-2-one 24 (0.155 mmol, 1 equiv.)and o-phenylenediamine (0.464 mmol, 3 equiv.)were added to a screwcapped reaction tube equipped with a magnetic stirring bar.Subsequently, glacial acetic acid (0.5 mL) was added and the mixture was stirred vigorously at 90 °C for 2-8 hour.The consumption of reactants and the formation of the product was followed by TLC (hexane/ethyl acetate).Distilled water was then added to the tube and the mixture was stirred at room temperature for 15 minutes.The precipitate was puried by column chromatography (silica gel, eluent hexane/ethyl acetate).In some cases, recrystallization was used for further purication aer doing column chromatography.
2.1.3.General procedure for the synthesis of pyrrolo[2,3-b] quinoxaline 3a 0 .1,5-Diphenyl-4-ethoxycarbonyl-3-hydroxy-3-pyrroline-2-one 24 (0.155 mmol, 1 equiv.),o-phenylenediamine (0.464 mmol, 3 equiv.)and citric acid (0.31 mmol, 2 equiv.)were added to a screw-capped reaction tube equipped with a magnetic stirring bar.Subsequently, absolute ethanol (1 mL) was added and the reaction mixture was stirred vigorously at 80 °C for 3 hours.Then, distilled water was added to the tube and the mixture was stirred at room temperature for 15 minutes.The precipitate was puried by column chromatography (silica gel, dichloromethane).; Ar-H), 4.32 (q, 3 J(H,H) = 7.14 Hz, 2H; OCH 2 ), 1.19 ppm (t, 3 J(H,H) = 7.14 Hz, 3H; CH 3 ). 136 DPPH solution (1 mM) was prepared in methanol and solutions of each pyrrolo[2,3-b]quinoxaline were prepared in DMSO at various concentrations (512, 265, 128, 32 and 8 mg mL −1 ).Then, 200 mL DPPH solution was added to 1.28 mL samples at each tested concentration and the free radical scavenging reactions were carried out on a 96well plate at 37 °C for 30 minutes.The absorbance was measured at 517 nm wavelength by a BioTek Epoch 2 Microplate Spectrophotometer.The percentage of free radical scavenging was calculated as SP (%) = [(OD 0 − OD 1 )/OD 0 ] × 100, where OD 0 was dened as the nal absorbance of the control reaction with quercetin as the reference antioxidant, and OD 1 stands for the absorbance in the presence of the sample.Each experiment was repeated three times and quercetin was used as the positive control.

Determination of crystal structure of 3a
Single crystals of 3a suitable for X-ray diffraction were obtained by evaporating a solution in dichloromethane/n-hexane mixture at room temperature.X-ray diffraction patterns were collected at 293(2) K on an Agilent SuperNova diffractometer, equipped with an Eos CCD detector, using Mo Ka radiation (l = 0.71073 Å).The images were interpreted and integrated with the CrysA-lisPro and the implemented absorption correction was applied. 27Using Olex2, 28 the structure was solved with the ShelXT 29 structure solution program using intrinsic phasing and rened with the ShelXL 30 renement package using fullmatrix least-squares minimization on F 2 .Non-hydrogen atoms were anisotropically rened and the hydrogen atoms in the riding mode with isotropic temperature factors were xed at 1.2 times U eq of the parent atoms (1.5 for methyl groups).Table S2 † gives crystal data and structure renement details for 3a.

Computational details
All DFT calculations were carried out with the Gaussian 09 suite of programs. 31M06-2X functional 32 and 6-311++G(d,p) basis set were used for all calculations.1][42][43] This method has been repeatedly benchmarked against experimental data, delivering results with low errors (k calc /k exp ratio = 1-2.9),particularly in lipid medium. 34,38,39As a reference all details of the calculations are shown in Table S1, ESI.†
The product 3a could be obtained in 83% yield when the concentration of aromatic amine 2 in the solvent increased to 0.93 M, while the amount of 3-pyrroline-2-one derivative 1a remained unchanged at 0.31 M. Therefore, the ratio 1 : 3.0 of  2).
The enol tautomer of 1,5-disubstituted-4-ethoxycarbonyl-3hydroxy-3-pyrroline-2-ones (1a-e) could be converted to the more stable keto form 1a 0 -e 0 in a polar solvent such as ethanol or acetic acid. 44The reactions between 1a-e/1a 0 -e 0 and o-phenylenediamine (2) are reversible and occur at the 3-position of pyrrolin-2,3-dione heterocyclic ring to yield imine intermediate 4 (ref.16) Intermediate 4 exists predominantly in the enamine form 5 due to resonance stabilization via intramolecular hydrogen bonding 45 and the intramolecular nucleophilic addition reaction between the amino (NH 2 ) and carbonyl group (C] O) in intermediate 5 results in products 3a 0 -e 0 .Finally, oxidative dehydrogenation of compounds 3a 0 -e 0 by oxygen molecules in fresh air will result in the formation of pyrrolo[2,3-b]quinoxalines 3a-e (Scheme 2). 46ll new compounds were characterized by NMR and high resolution mass spectrometry (HRMS).In the 1 H NMR spectrum of ethyl 1,2-diphenyl-2,4-dihydro-1H-pyrrolo[2,3-b]quinoxaline-3-carboxylate (3a 0 ), there is a characteristic broad and lowintensity peak corresponding to the proton of the secondary amino group (NH) at the chemical shi of 8.97 ppm.Undoubtedly, the intramolecular hydrogen bonding induced the higher chemical shi of the secondary amino proton.Furthermore, the proton at the 2-position of the pyrrole moiety of compound 3a 0 appears as a singlet at 6.24 ppm.In contrast, this peak disappears in the 1 H NMR spectrum of ethyl 1,2diphenyl-1H-pyrrolo[2,3-b]quinoxaline-3-carboxylate (3a).Moreover, 1 H NMR spectra of pyrrolo[2,3-b]quinoxalines 3a-e all showed two doublets of doublets (dd) at chemical shis around 8.41 ppm and 8.05 ppm, representing two protons at the 5,8-positions of the quinoxaline moieties.In addition, resonance signals of two methylene protons and three methyl protons of the ethoxycarbonyl group (-C(O)OCH 2 CH 3 ) in compounds 3a-e were characterized by a quartet (q) and a triplet (t) at chemical shis around 4.36 ppm and 1.26 ppm, respectively.

The radical scavenging activity of pyrrolo
(24% DPPH at 128 mg mL −1 ) exhibited the most effective DPPH antiradical activity.This performed over twofold better than 3b (DPPH: 7%) or 3d (DPPH: 9%).3c and 3e exhibited intermediate activities at 20% and 18% DPPH, respectively.Nevertheless, prior research has demonstrated that pyrrolo[2,3-b]quinoxaline derivatives possess signicant radical scavenging capabilities in contexts that are more biologically signicant, such as lipid peroxidation or against HOc radicals. 47-49Consequently, computational chemistry has been employed to further examine the radical (i.e., HOc and HOOc) scavenging activity of the most active compound (3a) in physiological environments.3.3.2.Calculations of the HOc/HOOc radical scavenging activity of the most active compound (3a) (a) Structure evaluation.The hexagon rings of 3a are capable of undergoing rotation in order to generate an extensive range of conformers.Since the conformer with the highest probability of engaging in a radical scavenging reaction is also the most stable, the initial step involved assessing the electron energy levels of every conceivable conformers of 3a. 50Following this, the conformer exhibiting the least amount of electronic energy was optimized utilizing the M06-2X/6-311++G (d,p) level of theory.The resulting data, including the angles (°) and bond length (Å), was subsequently compared to the crystal structure and is displayed in Fig. 3, S19 †, Table S2 and ESI.† It was found that the deviations in bond lengths and angles between the optimized and crystal structures were less than 0.025 Å and 2°, i.e. less than 1.7% and 1.6%, respectively.This internally conrms the high accuracy of the model chemistry used in this work.
(b) The antiradical activity in the gas phase.To identify the primary mechanisms by which 3a would function under the more complex physiological conditions, its antioxidant properties were initially evaluated in the gas phase.Depending on the molecular structures involved, various pathways may be followed by the 3a − HOc/HOOc reactions.2][53][54] To identify the preferred radical quenching mechanism (FHT, SET, and RAF) followed by 3a to eliminate the HOc and HOOc radicals, rst the gas phase Gibbs free energy changes (DG°) were computed.Table 4 displays the results.The radical scavenging activity for the HOc radicals are thermodynamically spontaneous (DG°< 0) at most positions in 3a, with the exception of the SET reaction (DG°= 155.6 kcal mol −1 ) and the RAF reactions at C4, C9, and N13 (DG°> 0).On the contrary, the radical scavenging of the HOOc radical is not spontaneous in any of the mechanisms that have been investigated.Thus, the radical scavenging of HOOc was excluded from the investigation.The kinetic evaluation of the possible positions (DG°< 0, Table 4) was conducted in a consistent manner to assess the scavenging of HOc radical by 3a in the gas phase.The outcomes of this analysis are displayed in Table 5, Fig. 4 and 5.
The results presented in the PES (Fig. 4) indicate that the C12 position has the lowest reaction barrier value of 3.6 kcal mol −1 .In contrast, the C4 position of 3a displayed the highest reaction barrier value of 15.0 kcal mol −1 .The FHT reaction of C23-H and the RAF at position C13 exhibited the second and third lowest reaction barriers, measuring 5.9 and 4.9 kcal mol −1 , respectively.In contrast, the 3a + HOc reaction has comparatively lower reaction barriers, ranging from 7.7 to 15.0 kcal mol −1 in the rest of the reactions.According to the PES analysis, the HOc  Paper RSC Advances radical scavenging activity of 3a is dominated by the FHT reaction at the C23-H bond and the RAF reaction at the C12/13.Table 5 indicates that the overall rate constant (k overall ) for the gas phase scavenging of the HOc radical was 2.70 × 10 11 M −1 s −1 .The rate constant for the 3a + HOc reaction was determined by the RAF reaction at positions C12 and C13, with rate constants of 1.45 × 10 11 (G = 53.5%)and 7.60 × 10 10 M −1 s −1 (G = 28.1%),respectively, while the H-abstraction contributed approximately 18.2% (k Eck = 4.94 × 10 10 M −1 s −1 ) to the overall rate constant.This result is consistent with the thermodynamic and the PES analysis (Table 4 and Fig. 4), which determined that the RAF-C12/13 and FHT-C23 reactions had the lowest DG°values (DG°= −20.7, −24.5 and −21.7 kcal mol −1 for C12, C13 and C23, respectively).No other reaction pathways make any notable contributions.
(c) The antiradical activity in the lipid medium.8][49] Therefore, the QM-ORSA protocol was employed here to assess the kinetics of the HOc-scavenging reactions taking place in the lipid medium following the main reactions in nonpolar media i.e., the RAF reaction at C12/13 and FHT reaction in the C23-H bond.The ndings are presented in Table 6.
Fig. 5 Optimized geometries of the main transition states between 3a and HOc radicals according to FHT and RAF processes.The k overall for the 3a + HOc reaction in pentyl ethanoate was 8.56 × 10 8 M −1 s −1 .The C12 position RAF mechanism (G = 51.4%) and the FHT pathway at the C23-H bond (G = 47.9%) were found to dene the HOc antiradical activity.In contrast, the C13 position RAF reaction accounted for a mere 0.7% of the overall reaction.The hydroxyl radical scavenging capability of 3a in a non-polar environment is analogous to that of widely used antioxidants including Trolox, melatonin, 55 indole-3carbinol, 56 and gallic acid. 573a is therefore an effective hydroxyl radical scavenger in lipid environments.

Conclusion
Successful synthesis of ve pyrrolo[2,3-b]quinoxaline derivatives was achieved.3a demonstrates the greatest DPPH radical scavenging activity, as determined by the DPPH assay.The structure of the synthesized lead compound 3a was veried by X-ray analysis.In pentyl ethanoate, the thermodynamic and kinetic calculations also revealed that 3a exhibited HO radical scavenging activity with k overall = 8.56 × 10 8 M −1 s −1 .In contrast, in nonpolar media, 3a demonstrated only a negligible capacity to scavenge hydroperoxyl radicals.The capacity of 3a to scavenge hydroxyl radicals in non-polar environments is comparable to that of conventional antioxidants including Trolox, melatonin, indole-3-carbinol, and gallic acid.Therefore in physiological lipid environments 3a holds potential as a HOc radical scavenger.

Fig. 2 A
Fig.2A view of the molecular structure of 3a, with atom labels and displacement ellipsoids drawn at the 30% probability level.H atoms are shown as small circles of arbitrary radii.

Table 4
Calculated DG°values (in kcal mol −1 ) of the reactions of 3a with HOc following the FHT and RAF mechanisms

Table 6
The calculated DG ‡ (kcal mol −1 ), k app , k overall (M −1 s −1 ) and G (%) at 298.15 K, in the 3a oxidation by HOc radicals in the lipid medium